Background
The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus and resulting COVID‐19 pandemic present important diagnostic challenges. Several diagnostic strategies are available ...to identify current infection, rule out infection, identify people in need of care escalation, or to test for past infection and immune response. Serology tests to detect the presence of antibodies to SARS‐CoV‐2 aim to identify previous SARS‐CoV‐2 infection, and may help to confirm the presence of current infection.
Objectives
To assess the diagnostic accuracy of antibody tests to determine if a person presenting in the community or in primary or secondary care has SARS‐CoV‐2 infection, or has previously had SARS‐CoV‐2 infection, and the accuracy of antibody tests for use in seroprevalence surveys.
Search methods
We undertook electronic searches in the Cochrane COVID‐19 Study Register and the COVID‐19 Living Evidence Database from the University of Bern, which is updated daily with published articles from PubMed and Embase and with preprints from medRxiv and bioRxiv. In addition, we checked repositories of COVID‐19 publications. We did not apply any language restrictions. We conducted searches for this review iteration up to 27 April 2020.
Selection criteria
We included test accuracy studies of any design that evaluated antibody tests (including enzyme‐linked immunosorbent assays, chemiluminescence immunoassays, and lateral flow assays) in people suspected of current or previous SARS‐CoV‐2 infection, or where tests were used to screen for infection. We also included studies of people either known to have, or not to have SARS‐CoV‐2 infection. We included all reference standards to define the presence or absence of SARS‐CoV‐2 (including reverse transcription polymerase chain reaction tests (RT‐PCR) and clinical diagnostic criteria).
Data collection and analysis
We assessed possible bias and applicability of the studies using the QUADAS‐2 tool. We extracted 2x2 contingency table data and present sensitivity and specificity for each antibody (or combination of antibodies) using paired forest plots. We pooled data using random‐effects logistic regression where appropriate, stratifying by time since post‐symptom onset. We tabulated available data by test manufacturer. We have presented uncertainty in estimates of sensitivity and specificity using 95% confidence intervals (CIs).
Main results
We included 57 publications reporting on a total of 54 study cohorts with 15,976 samples, of which 8526 were from cases of SARS‐CoV‐2 infection. Studies were conducted in Asia (n = 38), Europe (n = 15), and the USA and China (n = 1). We identified data from 25 commercial tests and numerous in‐house assays, a small fraction of the 279 antibody assays listed by the Foundation for Innovative Diagnostics. More than half (n = 28) of the studies included were only available as preprints.
We had concerns about risk of bias and applicability. Common issues were use of multi‐group designs (n = 29), inclusion of only COVID‐19 cases (n = 19), lack of blinding of the index test (n = 49) and reference standard (n = 29), differential verification (n = 22), and the lack of clarity about participant numbers, characteristics and study exclusions (n = 47). Most studies (n = 44) only included people hospitalised due to suspected or confirmed COVID‐19 infection. There were no studies exclusively in asymptomatic participants. Two‐thirds of the studies (n = 33) defined COVID‐19 cases based on RT‐PCR results alone, ignoring the potential for false‐negative RT‐PCR results. We observed evidence of selective publication of study findings through omission of the identity of tests (n = 5).
We observed substantial heterogeneity in sensitivities of IgA, IgM and IgG antibodies, or combinations thereof, for results aggregated across different time periods post‐symptom onset (range 0% to 100% for all target antibodies). We thus based the main results of the review on the 38 studies that stratified results by time since symptom onset. The numbers of individuals contributing data within each study each week are small and are usually not based on tracking the same groups of patients over time.
Pooled results for IgG, IgM, IgA, total antibodies and IgG/IgM all showed low sensitivity during the first week since onset of symptoms (all less than 30.1%), rising in the second week and reaching their highest values in the third week. The combination of IgG/IgM had a sensitivity of 30.1% (95% CI 21.4 to 40.7) for 1 to 7 days, 72.2% (95% CI 63.5 to 79.5) for 8 to 14 days, 91.4% (95% CI 87.0 to 94.4) for 15 to 21 days. Estimates of accuracy beyond three weeks are based on smaller sample sizes and fewer studies. For 21 to 35 days, pooled sensitivities for IgG/IgM were 96.0% (95% CI 90.6 to 98.3). There are insufficient studies to estimate sensitivity of tests beyond 35 days post‐symptom onset. Summary specificities (provided in 35 studies) exceeded 98% for all target antibodies with confidence intervals no more than 2 percentage points wide. False‐positive results were more common where COVID‐19 had been suspected and ruled out, but numbers were small and the difference was within the range expected by chance.
Assuming a prevalence of 50%, a value considered possible in healthcare workers who have suffered respiratory symptoms, we would anticipate that 43 (28 to 65) would be missed and 7 (3 to 14) would be falsely positive in 1000 people undergoing IgG/IgM testing at days 15 to 21 post‐symptom onset. At a prevalence of 20%, a likely value in surveys in high‐risk settings, 17 (11 to 26) would be missed per 1000 people tested and 10 (5 to 22) would be falsely positive. At a lower prevalence of 5%, a likely value in national surveys, 4 (3 to 7) would be missed per 1000 tested, and 12 (6 to 27) would be falsely positive.
Analyses showed small differences in sensitivity between assay type, but methodological concerns and sparse data prevent comparisons between test brands.
Authors' conclusions
The sensitivity of antibody tests is too low in the first week since symptom onset to have a primary role for the diagnosis of COVID‐19, but they may still have a role complementing other testing in individuals presenting later, when RT‐PCR tests are negative, or are not done. Antibody tests are likely to have a useful role for detecting previous SARS‐CoV‐2 infection if used 15 or more days after the onset of symptoms. However, the duration of antibody rises is currently unknown, and we found very little data beyond 35 days post‐symptom onset. We are therefore uncertain about the utility of these tests for seroprevalence surveys for public health management purposes. Concerns about high risk of bias and applicability make it likely that the accuracy of tests when used in clinical care will be lower than reported in the included studies. Sensitivity has mainly been evaluated in hospitalised patients, so it is unclear whether the tests are able to detect lower antibody levels likely seen with milder and asymptomatic COVID‐19 disease.
The design, execution and reporting of studies of the accuracy of COVID‐19 tests requires considerable improvement. Studies must report data on sensitivity disaggregated by time since onset of symptoms. COVID‐19‐positive cases who are RT‐PCR‐negative should be included as well as those confirmed RT‐PCR, in accordance with the World Health Organization (WHO) and China National Health Commission of the People's Republic of China (CDC) case definitions. We were only able to obtain data from a small proportion of available tests, and action is needed to ensure that all results of test evaluations are available in the public domain to prevent selective reporting. This is a fast‐moving field and we plan ongoing updates of this living systematic review.
The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely ...known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to ...detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.
The rapidly spreading outbreak of COVID-19 disease is caused by the SARS-CoV-2 virus, first reported in December 2019 in Wuhan, China. As of June 17, 2020, this virus has infected over 8.2 million ...people but ranges in symptom severity, making it difficult to assess its overall infection rate. There is a need for rapid and accurate diagnostics to better monitor and prevent the spread of COVID-19. In this review, we present and evaluate two main types of diagnostics with FDA-EUA status for COVID-19: nucleic acid testing for detection of SARS-CoV-2 RNA, and serological assays for detection of SARS-CoV-2 specific IgG and IgM patient antibodies, along with the necessary sample preparation for accurate diagnoses. In particular, we cover and compare tests such as the CDC 2019-nCoV RT-PCR Diagnostic Panel, Cellex's qSARS-CoV-2 IgG/IgM Rapid Test, and point-of-care tests such as Abbott's ID NOW COVID-19 Test. Antibody testing is especially important in understanding the prevalence of the virus in the community and to identify those who have gained immunity. We conclude by highlighting the future of COVID-19 diagnostics, which include the need for quantitative testing and the development of emerging biosensors as point-of-care tests.
•Current standard-of-care for COVID-19 diagnosis is RT-PCR, used to detect SARS-CoV-2 viral RNA.•Serological assays for detection of SARS-CoV-2 specific IgG and IgM antibodies are useful to assess virus prevalence.•Point-of-care tests for COVID-19 diagnoses can expedite results and alleviate the burden placed on healthcare providers.•Future testing strategies include developing quantitative COVID-19 diagnostic tools.
•Chronic neurological disease and acute abnormalities are present in COVID-19 brain autopsies.•Acute hypoxic-injury, hemorrhage, and minimal inflammation are frequently observed.•Low levels of viral ...SARS-CoV-2 RNA are present; cellular source remains unknown.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) for which there have been over 50 million confirmed cases and 1.2 million deaths globally. While many SARS-CoV-2 infected individuals are asymptomatic or experience respiratory symptoms, extrapulmonary manifestations, including neurological symptoms and conditions, are increasingly recognized. There remains no clear understanding of the mechanisms that underlie neurological symptoms in COVID-19 and whether SARS-CoV-2 has the potential for neuroinvasion in humans. In this minireview, we discuss what is known from human autopsies in fatal COVID-19, including highlighting studies that investigate for the presence of SARS-CoV-2 in brain and olfactory tissue, and summarize the neuropathological consequences of infection. Incorporating microscopic and molecular findings from brain tissue into what we know about clinical disease will inform best practice management guidance and direct research priorities as it relates to neurological morbidity from COVID-19.
Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data ...normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.
Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. ...This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.
•Integrated CRISPR for COVID-19 clinical diagnosis.•Rapidly identify SARS-CoV-2-specific RNA signatures at an ultra-low concentration.•Detection environment friendly and compatible for high throughput COVID-19 screening in hospital.•Successful development and validation with swab samples from patients.•Improve diagnosis of individuals with suspected COVID-19 infections.
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing ...has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.
The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health ...interventions. Currently, several quantitative reverse transcription-PCR (RT-qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR analytical efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charité) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.
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